The increasing prevalence of multidrug-resistant pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) underscores the need to develop novel strategies to treat life-threatening infections. We plan to develop an integrated platform to discover new pathways for antibacterials, new compounds to inhibit those pathways, rapid methods to identify molecular targets of promising compounds, and rational strategies to select compound-target pairs for further development. The platform has three aims focusing on S. aureus but can be extended to other pathogens. The objective ofthe Aim 1 is to identify cell envelope pathways that can be targeted by antibacterials. We are focusing on the cell envelope because defects in envelope integrity are frequently lethal in invivo. We will probe highly saturated transposon libraries with small molecules that have cell envelope targets and use Tn-seq to identify genes that drop out ofthe libraries. We will validate that these genes interact with the cell envelope targets being inhibited by the small molecules. With the Discovery & Translational Services (DTS) Core, we will analyze the fitness of validated mutant strains in animals to prioritize targets. The objective of the Aim 2 is to identify compounds that inhibit high priority targets (HPTs). We will use a novel cell-based, pathway-directed screening paradigm in which a panel of mutant strains are screened against the same compound library and compared to the wild type parent strain. One mutant strain will have a deletion in HPT while the others will have deletions in genes that have a synthetic lethal interaction with the HPT. Hits against the HPT will be identified as compounds that do not affect the growth of the wild type or AHPT strain, but inhibit growth in the other mutant strains. By using cell based pathway direct screens, we envision greatly shortening the lag between identifying possible antibacterial targets and discovering compounds that inhibit them. The objective of Aim 3 is to establish a strategy to define and select compound-target pairs for further development We will develop a phage-based transposition method to identify the specific targets of confirmed hits (from Aim 2) within the genetic interaction networks (defined in Aim 1). We will generate a pool of mutants that over-express, under-express, or have genes deleted across the entire genome, from which the specific molecular target can be inferred. Target mutations conferring resistance will be evaluated by comprehensive protein mutagenesis, and the most promising compound-target pairs will advance into antibacterial lead optimization with the DTS Core.